Article

Generation of Transgene-Free iPSC Lines from Human Normal and Neoplastic Blood Cells Using Episomal Vectors.

Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2013; 997:163-76. DOI: 10.1007/978-1-62703-348-0_13
Source: PubMed

ABSTRACT Human induced pluripotent stem cells (iPSCs) have become an important tool for modeling human diseases and are considered a potential source of therapeutic cells. Original methods for iPSC generation use fibroblasts as a cell source for reprogramming and retroviral vectors as a delivery method of the reprogramming factors. However, fibroblasts require extended time for expansion and viral delivery of transgenes results in the integration of vector sequences into the genome which is a source of potential insertion mutagenesis, residual expressions, and reactivation of transgenes during differentiation. Here, we provide a detailed protocol for the efficient generation of transgene-free iPSC lines from human bone marrow and cord blood cells with a single transfection of non-integrating episomal plasmids. This method uses mononuclear bone marrow and cord blood cells, and makes it possible to generate transgene-free iPSCs 1-3 weeks faster than previous methods of reprogramming with fibroblasts. Additionally, we show that this approach can be used for efficient reprogramming of chronic myeloid leukemia cells.

Download full-text

Full-text

Available from: Kejin Hu, Jul 19, 2014
0 Followers
 · 
99 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human induced pluripotent stem cells (hiPSCs) are derived from a direct reprogramming of human somatic cells to a pluripotent stage through ectopic expression of specific transcription factors. These cells have two important properties, which are the self-renewal capacity and the ability to differentiate into any cell type of the human body. So, the discovery of hiPSCs opens new opportunities in biomedical sciences, since these cells may be useful for understanding the mechanisms of diseases in the production of new diseases models, in drug development/drug toxicity tests, gene therapies, and cell replacement therapies. However, the hiPSCs technology has limitations including the potential for the development of genetic and epigenetic abnormalities leading to tumorigenicity. Nowadays, basic research in the hiPSCs field has made progress in the application of new strategies with the aim to enable an efficient production of high-quality of hiPSCs for safety and efficacy, necessary to the future application for clinical practice. In this review, we show the recent advances in hiPSCs' basic research and some potential clinical applications focusing on cancer. We also present the importance of the use of statistical methods to evaluate the possible validation for the hiPSCs for future therapeutic use toward personalized cell therapies.
    10/2013; 2013:430290. DOI:10.1155/2013/430290
  • [Show abstract] [Hide abstract]
    ABSTRACT: Myeloid phagocytes (neutrophils, monocytes, macrophages, dendritic cells) have key roles in immune defense, as well as in tissue repair and remodeling. Defective or dysregulated myeloid phagocyte production or function can cause immune dysfunction, blood cell malignancies and inflammatory diseases. The tumor microenvironment can also condition myeloid phagocytes to promote tumor growth. Studies of their physiological and pathophysiological roles and the mechanisms regulating their production and function are crucial for the identification of novel therapeutic targets. In this review, we examine the use of induced pluripotent stem cells to study myeloid phagocytes in human diseases and develop future therapeutic strategies.
    Drug discovery today 01/2014; 19(6). DOI:10.1016/j.drudis.2014.01.004
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Generation of induced pluripotent stem cells (iPSC) via the ectopic expression of reprogramming factors is a simple, advanced, yet often perplexing technology due to low efficiency, slow kinetics and the use of numerous distinct systems for factor delivery. Scientists have used almost all available approaches for the delivery of reprogramming factors. Even the well-established retroviral vectors confuse some scientists due to different tropisms in use. The canonical virus-based reprogramming poses many problems including insertional mutagenesis, residual expression and re-activation of reprogramming factors, uncontrolled silencing of transgenes, apoptosis, cell senescence and strong immunogenicity. To eliminate or alleviate these problems, scientists have tried various other approaches for factor delivery and transgene removal. These include transient transfection, non-integrating viral vectors, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, miRNA transfection, RNA virion, RNA replicon, non-integrating replicating episomal plasmids, minicircles, polycistron and pre-integration of inducible reprogramming factors. These alternative approaches have their own limitations. Even iPSCs generated with RNA approaches should be screened for possible transgene insertions mediated by active endogenous retroviruses in the human genome. Even experienced researchers may encounter difficulty in selecting and using these different technologies. This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.
    Stem cells and development 02/2014; DOI:10.1089/scd.2013.0620